Evaluation of the effects of clearing agents, fixation, and process durations on cardiovascular tissue imaging with second harmonic generation and multi-photon modalities

Maedeh Makki¹Zachary A Molander¹Sergio A Pineda-Castillo²Devin W Laurence³Shubhra Singhal¹Yasmin Eltwafsha¹Gerhard A. Holzapfel⁴Tingting Gu^5*Chung-Hao Lee^1*

• ¹University of California, Riverside, Riverside, United States
• ²University of Minnesota Health Sciences, University of Minnesota Medical Center, Minneapolis, Minnesota, United States
• ³Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, United States
• ⁴Graz University of Technology, Graz, Styria, Austria
• ⁵University of Oklahoma, Norman, Oklahoma, United States

Protocols for tissue clearing have been established and optimized for the central nervous system. However, significant modifications are required for clearing different tissue types. Therefore, effective optical clearing for cardiovascular tissue remains a major challenge. The goal of this study is to better understand the responses of porcine left anterior descending artery (LADA) to label-free multiphoton imaging. To this end, the effects of different clearing methods (i.e., benzyl alcohol benzyl benzoate -BABB and glycerol), formalin fixation, variations in formalin fixation times, and extended storage in BABB are investigated. We compare tissue characteristics under different conditions (e.g., tissue clearing reagent and/or tissue fixation), particularly with regard to tissue preservation and transparency across z-stacks (i.e., imaging depths). The glycerol clearing method exhibited relatively lower tissue transparency, whereas the BABB clearing method significantly improved imaging clarity and enabled robust signal intensities at deeper layers of LADA tissue. In addition, we observed that BABB preserves fluorescent signals even after extended tissue storage. Finally, we found that formalin fixation in combination with the glycerol clearing method significantly improved tissue preservation compared to the glycerol clearing method alone. However, in combination with the BABB clearing method, fixation reduced tissue transparency and signal intensity compared to BABB clearing without fixation.